EP2945962B1 - Verfahren zur isolierung von blutprodukten aus einem inter-alpha-inhibitorprotein-abgereicherten blutproduktmaterial - Google Patents

Verfahren zur isolierung von blutprodukten aus einem inter-alpha-inhibitorprotein-abgereicherten blutproduktmaterial Download PDF

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EP2945962B1
EP2945962B1 EP14740523.7A EP14740523A EP2945962B1 EP 2945962 B1 EP2945962 B1 EP 2945962B1 EP 14740523 A EP14740523 A EP 14740523A EP 2945962 B1 EP2945962 B1 EP 2945962B1
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factor
iαip
product material
blood product
igg
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EP2945962A4 (de
EP2945962A1 (de
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Yow-Pin Lim
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Prothera Biologics Inc
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Prothera Biologics Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/36—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving ionic interaction, e.g. ion-exchange, ion-pair, ion-suppression or ion-exclusion
    • B01D15/361—Ion-exchange
    • B01D15/363—Anion-exchange
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/18—Ion-exchange chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/745—Blood coagulation or fibrinolysis factors
    • C07K14/75—Fibrinogen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/745—Blood coagulation or fibrinolysis factors
    • C07K14/755—Factors VIII, e.g. factor VIII C (AHF), factor VIII Ag (VWF)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/76—Albumins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
    • C07K14/8121—Serpins
    • C07K14/8125—Alpha-1-antitrypsin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6456—Plasminogen activators
    • C12N9/6462—Plasminogen activators u-Plasminogen activator (3.4.21.73), i.e. urokinase

Definitions

  • I ⁇ Ip inter-alpha inhibitor proteins
  • albumin immunoglobulins
  • IVIg immunoglobulins
  • factor VII, factor VIII, factor IX alpha-1 antitrypsin
  • alpha-1 antitrypsin anti-thrombin III
  • C1-inhibitor protein C
  • von Willebrand factor factor H
  • prothrombin factor II
  • thrombin thrombin
  • Blood products serve key roles in clotting, immunity, inflammation, and other biological functions.
  • Subjects deficient in one or more of these compounds may suffer from a variety of medical conditions; treatment with particular blood compounds may alleviate these conditions or their symptoms. Further, treatment with particular blood products can produce medical benefits, such as the prevention of sepsis or neuronal damage.
  • the efficient purification of blood compounds is of particular importance in light of the limited supply of blood for this purpose.
  • Isolation of multiple blood products from a single starting material maximizes the efficiency of blood product isolation.
  • the present invention provides such a method.
  • one or more blood products are isolated from a blood product material previously depleted of one or more inter-alpha inhibitor proteins (I ⁇ Ip).
  • I ⁇ Ip inter-alpha inhibitor proteins
  • the present invention provides a method for isolating one or more blood products from an inter-alpha inhibitor protein (I ⁇ Ip)-depleted blood product material.
  • the method includes providing an I ⁇ Ip-depleted blood product material that is depleted of one or more I ⁇ Ip family members by at least about 20% of the total present in the source blood product material and that includes at least about 20% of IgG present in the source blood product material.
  • the method further includes isolating one or more blood products from the I ⁇ Ip-depleted blood product material, at least one of which is selected from albumin, IgA, IgG, IgM, IgD, IgG, IVIg, anti-D IgG, hepatitis B IgG, measles IgG, rabies IgG, tetanus IgG, Varicella Zoster IgG, fibrinogen (factor I), prothrombin (factor II), thrombin, anti-thrombin III, factor III, factor V, VII, factor VIII, factor IX, factor X, factor XI, factor XII, factor XIII, fibronectin, alpha-1 antitrypsin, alpha-2 antiplasmin, urokinase, C1-inhibitor, protein C, protein S, protein Z, protein Z-related protease inhibitor, plasminogen, tissue plasminogen activator, plasminogen activator inhibitor-1,
  • the I ⁇ Ip-depleted blood product material substantially includes 3 or more non-lalp blood products, substantially includes 10 or more non-lalp blood products, or substantially includes 20 or more non-lalp blood products selected from albumin, IgA, IgG, IgM, IgD, IgG, IVIg, anti-D IgG, hepatitis B IgG, measles IgG, rabies IgG, tetanus IgG, Varicella Zoster IgG, fibrinogen (factor I), prothrombin (factor II), thrombin, anti-thrombin III, factor III, factor V, factor VII, factor VIII, factor IX, factor X, factor XI, factor XII, factor XIII, fibronectin, alpha-1 antitrypsin, alpha-2 antiplasmin, urokinase, C1-inhibitor, protein C, protein S, protein Z, protein Z-related protease inhibitor,
  • the isolating step includes the steps of contacting the I ⁇ Ip-depleted blood product material to a support such that one or more of the blood products is substantially retained on the support, and subsequently eluting from the support a fraction enriched in at least one of the substantially retained blood products.
  • the support is a chromatography column.
  • the I ⁇ Ip-depleted blood product material may be a material depleted of one or more (e.g., two, three, four, or more) I ⁇ Ip family members, such as I ⁇ I, P ⁇ I, and/or bikunin, or both I ⁇ I and P ⁇ I; the depletion of the one or more I ⁇ Ip family members is by at least about 20% or by at least about 90% or more.
  • I ⁇ Ip family members such as I ⁇ I, P ⁇ I, and/or bikunin, or both I ⁇ I and P ⁇ I
  • the depletion of the one or more I ⁇ Ip family members is by at least about 20% or by at least about 90% or more.
  • the method for isolating one or more blood products from an I ⁇ Ip-depleted blood product material includes contacting to a first support a blood product material that includes at least I ⁇ Ip, IgG in an I ⁇ Ip family:IgG weight ratio equal to about 1:30, equal to about 1:5, or between about 1:30 and about 1:5, and one of factor VIII in a factor VIII:I ⁇ Ip family weight ratio equal to or less than about 1:10 6 and von Willebrand factor in a von Willebrand factor:I ⁇ Ip family weight ratio equal to or less than about 1:40, by which I ⁇ Ip is substantially retained on the first support and material not retained by the support is included in a first flow-through.
  • This aspect further includes isolating at least one of the one or more blood products from the first flow-through, at least one of which is selected from albumin, IgA, IgG, IgM, IgD, IgG, IVIg, anti-D IgG, hepatitis B IgG, measles IgG, rabies IgG, tetanus IgG, Varicella Zoster IgG, fibrinogen (factor I), prothrombin (factor II), thrombin, anti-thrombin III, factor III, factor V, VII, factor VIII, factor IX, factor X, factor XI, factor XII, factor XIII, fibronectin, alpha-1 antitrypsin, alpha-2 antiplasmin, urokinase, C1-inhibitor, protein C, protein S, protein Z, protein Z-related protease inhibitor, plasminogen, tissue plasminogen activator, plasminogen activator inhibitor-1, plasmin
  • the blood product material is whole plasma, cryo-poor plasma, liquid plasma, fresh frozen plasma (FFP), FFP24, frozen plasma (FP), FP24, thawed FFP, thawed FFP24, thawed FP, thawed FP24, source plasma, recovered plasma, solvent/detergent-treated plasma (SDP), platelet-rich plasma (PRP), platelet-poor plasma (PPP), serum, blood, or a diluted or concentrated preparation thereof.
  • the blood product material may be admixed with loading buffer prior to contacting the first support.
  • the loading buffer includes 10 to 300 mM salt, such as 10, 20, 30, 40, 50, 100, 150, 200, 250, or 300 mM salt. In certain embodiments, the loading buffer includes 50 to 250 mM salt, such as 50, 60, 70, 80, 90, 100, 150,200, or 250 mM salt.
  • the support is a QA support and the loading buffer includes about 10 to 100 mM salt, such as 10, 20, 30, 40, 50, 60, 75, 80, 85, 90, 95, or 100 mM salt.
  • the pH is from 5.5 to 6.5, such as 5.5, 5.7, 5.9, 6.0, 6.1, 6.3, or 6.5.
  • the support is a QA support, the loading buffer includes about 50 mM salt, and the pH is about 6.0.
  • the support is a DEAE support and the loading buffer includes about 150 to 250 mM salt, such as 150, 175, 200, 225, or 250 mM salt.
  • the pH is from 7.0 to 8.0, such as 7.0, 7.2, 7.4, 7.6, 7.8, or 8.0.
  • the support is a DEAE support, the loading buffer includes about 200 mM salt, and the pH is about 7.6.
  • the first flow-through includes three or more non-lalp blood products, 10 or more non-lalp blood products, or 20 or more non-lalp blood products in an amount equal to or greater than about 20% of the amount present in the blood product material, and wherein each of said non-lalp blood products is selected from albumin, IgA, IgG, IgM, IgD, IgG, IVIg, anti-D IgG, hepatitis B IgG, measles IgG, rabies IgG, tetanus IgG, Varicella Zoster IgG, fibrinogen (factor I), prothrombin (factor II), thrombin, anti-thrombin III, factor III, factor V, factor VII, factor VIII, factor IX, factor X, factor XI, factor XII, factor XIII, fibronectin, alpha-1 antitrypsin, alpha-2 antiplasmin, urokinase, C1-inhibi
  • the isolating step includes contacting the first flow-through to a second support such that one or more of the blood products is substantially retained on the second support, and subsequently eluting from the second support a fraction enriched in at least one of the substantially retained blood products.
  • one or both of the first or second supports is a chromatography column.
  • the first support may be an anion exchange column.
  • the first support may be a DEAE or QA column.
  • Further embodiments include eluting the substantially retained I ⁇ Ip from the first support, producing a first eluate that is enriched with I ⁇ Ip.
  • the first eluate may consist of isolated I ⁇ Ip.
  • the method further includes isolating the substantially retained I ⁇ Ip from the first eluate.
  • the yield of the isolated I ⁇ Ip may be at least 5 ⁇ g/ml blood product material, at least 50 ⁇ g/ml blood product material, at least 100 ⁇ g/ml blood product material, at least 300 ⁇ g/ml blood product material, at least 600 ⁇ g/ml blood product material, or at least 900 ⁇ g/ml blood product material.
  • the purity of isolated I ⁇ Ip may be at least 5%, at least 25%, at least 50%, is at least 75%, or 100%.
  • the isolated I ⁇ Ip may be I ⁇ I, P ⁇ I, or bikunin, or may include two or more I ⁇ Ip family members, or may include I ⁇ I and P ⁇ I.
  • the substantially retained I ⁇ Ip may be I ⁇ I, P ⁇ I, or bikunin, or may include two or more I ⁇ Ip family members, or may include I ⁇ I and P ⁇ I.
  • the yield of one or more isolated non-lalp blood products may be at least 20% of the total present in the first flow-through, at least 50% of the total present in the first flow-through, or at least 80% of the total present in the first flow-through.
  • the I ⁇ Ip-depleted blood product material is a blood product material depleted of I ⁇ Ip (e.g., one or more (e.g., two, three, four, or more) I ⁇ Ip family members, such as I ⁇ I, P ⁇ I, and/or bikunin, or both I ⁇ I and P ⁇ I) by at least about 20% (e.g., by at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% or more, or a depletion within the range of 20%-90%) of the total I ⁇ Ip present in the source blood product material.
  • I ⁇ Ip e.g., one or more (e.g., two, three, four, or more) I ⁇ Ip family members, such as I ⁇ I, P ⁇ I, and/or bikunin, or both I ⁇ I and P ⁇ I) by at least about 20% (e.g., by at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%
  • the I ⁇ Ip-depleted blood product material substantially includes 3 or more non-lalp blood products, substantially includes 10 or more non-lalp blood products, or substantially includes 20 or more non-lalp blood products selected from albumin, IgA, IgG, IgM, IgD, IgG, IVIg, anti-D IgG, hepatitis B IgG, measles IgG, rabies IgG, tetanus IgG, Varicella Zoster IgG, fibrinogen (factor I), prothrombin (factor II), thrombin, anti-thrombin III, factor III, factor V, factor VII, factor VIII, factor IX, factor X, factor XI, factor XII, factor XIII, fibronectin, alpha-1 antitrypsin, alpha-2 antiplasmin, urokinase, C1-inhibitor, protein C, protein S, protein Z, protein Z-related protease inhibitor,
  • the invention is directed to a method for isolating one or more blood products from an inter-alpha inhibitor protein (I ⁇ Ip)-depleted blood product material, comprising:
  • Blood product means a commercially valuable substance that may be naturally present in blood, for example in the blood of a human, and for which isolation from blood is commercially practiced.
  • Blood products include I ⁇ Ip, albumin, IgA, IgG, IgM, IgD, IgG, IVIg, anti-D IgG, hepatitis B IgG, measles IgG, rabies IgG, tetanus IgG, Varicella Zoster IgG, fibrinogen (factor I), prothrombin (factor II), thrombin, anti-thrombin III, factor III, factor V, factor VII, factor VIII, factor IX, factor X, factor XI, factor XII, factor XIII, fibronectin, alpha-1 antitrypsin, alpha-2 antiplasmin, urokinase, C1-inhibitor, protein C, protein S, protein Z, protein Z-related protease inhibitor, plasminogen,
  • I ⁇ Ip means a blood product composed of one or more or all members of the inter-alpha inhibitor protein (I ⁇ Ip) family, each member being composed of a light chain, also called bikunin, optionally linked to one or more heavy chains (e.g., heavy chains H1, H2, H3, and/or H4).
  • I ⁇ Ip inter-alpha inhibitor protein
  • I ⁇ Ip family examples include inter-alpha inhibitor (I ⁇ I) composed of bikunin linked to 2 heavy polypeptide chains (e.g., both H1, both H2, or H1 and H2) and having a molecular weight of about 225 to about 260 kDA and pre-alpha inhibitor (P ⁇ I) composed of bikunin linked to a single heavy chain (e.g., H1, H2, H3, or H4) and having a molecular weight of about 110 to about 130 kDA.
  • I ⁇ Ip may be bikunin alone and/or the combination of bikunin with one or more heavy chains.
  • I ⁇ Ip family means all members of the I ⁇ Ip family.”
  • Blood product material means any blood-derived composition that includes at least I ⁇ Ip and IgG in an I ⁇ Ip family:IgG weight ratio equal to about 1:30, equal to about 1:5, or between about 1:30 and about 1:5, such as about 1:30, 1:25, 1:20, 1:15, 1:10, or 1:5, and one of factor VIII in a factor VIII:I ⁇ Ip family weight ratio equal to or less than about 1:10 6 , such as about 1:10 6 , 1:10 7 , 1:10 8 , 1:10 9 , or 1:10 10 , and von Willebrand factor in a von Willebrand factor:I ⁇ Ip family weight ratio equal to or less than about 1:40, such as about 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, or 1:200.
  • a blood product material may be, for example, whole plasma, cryo-poor plasma, liquid plasma, thawed fresh frozen plasma (FFP), thawed FFP24, thawed frozen plasma (FP), thawed FP24, source plasma, recovered plasma, solvent/detergent-treated plasma (SDP), platelet-rich plasma (PRP), platelet-poor plasma (PPP), serum, blood, or a diluted or concentrated preparation thereof.
  • FFP fresh frozen plasma
  • FP24 thawed frozen plasma
  • FP thawed frozen plasma
  • SDP solvent/detergent-treated plasma
  • PRP platelet-rich plasma
  • PPP platelet-poor plasma
  • serum blood, or a diluted or concentrated preparation thereof.
  • Support means any apparatus that interacts with at least one blood product in a manner that is dependent upon the properties of the blood product, such that the apparatus is useful in fractionating the blood products present in a mixture or solution.
  • a support may be a column, a membrane, a disc, a chip, or other apparatus for chromatography or affinity capture, examples of which are known in the art.
  • Substantially retained means at least about 20%, such as about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%, of the amount of a substance present in a starting material is captured on a support.
  • To “deplete” means to reduce the concentration or amount of a substance.
  • the concentration or amount of a substance may be considered depleted if it is reduced by about 20% to about 100%, such as about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.
  • I ⁇ Ip-depleted blood product material means any material derived from a blood product material that is depleted of one or more or all I ⁇ Ip family members and further includes IgG. More specifically, an I ⁇ Ip-depleted blood product material includes no more than about 80%, such as about 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 1%, 0.1%, 0.01% or 0%, of the total of one or more or all lalp family members and at least about 20%, such as about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%, of total IgG present in the starting blood product material. An I ⁇ Ip-depleted blood product material may also include one or more additional blood products.
  • Isolated means to have separated about 20% to 100%, such as about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%, of a blood product from a starting material.
  • a starting material may be a blood product material, an I ⁇ Ip-depleted blood product material, or an I ⁇ Ip-depleted blood product material having been further depleted of one or more non-lalp blood products.
  • Purity means the extent to which a blood product that has been isolated, such as a blood product isolated by the methods of the present invention, is free of other components. Purity is expressed as the percentage by weight of blood product in an isolated blood product composition.
  • Blood product materials may contain numerous blood products, the isolation of some or all of which may be of medical or economic value.
  • the present invention is directed toward a method of sequentially isolating multiple blood components from a single starting sample. More specifically, the discovery of the present invention is directed to methods of isolating one or more blood products from an I ⁇ Ip-depleted blood product material.
  • the I ⁇ Ip family is a group of structurally related plasma-associated serine protease inhibitors. Members of this family are composed of heavy and light polypeptide subunits that are covalently linked by a glycosaminoglycan.
  • the light chain also called bikunin, is responsible for the serine protease inhibitory activity of inter-alpha proteins.
  • the name "bikunin” reflects the presence of two protease-inhibiting domains of the Kunitz type.
  • the heavy chains of inter-alpha proteins (H1, H2, H3, H4) are also called Hyaluronic acid (HA) binding proteins.
  • bikunin In normal plasma, bikunin is found mostly in a complex form as inter-alpha inhibitor (I ⁇ I), which has a molecular weight of about 225 to about 260 kDa, and pre-alpha inhibitor (P ⁇ I), which has molecular weight of about 110 to about 130 kDa.
  • I ⁇ I inter-alpha inhibitor
  • P ⁇ I pre-alpha inhibitor
  • I ⁇ I bikunin is linked to 2 heavy polypeptide chains (e.g., H1 and H2), whereas, in P ⁇ I, only a single heavy chain (e.g., H3) is linked to bikunin.
  • the I ⁇ Ip-depleted blood product material may be produced by the use of a support to isolate I ⁇ Ip (e.g., one or more (e.g., two, three, four, or more, or all) I ⁇ Ip family members, such as I ⁇ I, P ⁇ I, and/or bikunin, or both I ⁇ I and P ⁇ I) from a blood product material.
  • I ⁇ Ip may be isolated from a blood product material by any applicable method known in the art, including methods of chromatography, such as anion exchange chromatography, cation exchange chromatography, affinity chromatography, or dye-ligand chromatography.
  • Exemplary methods for the isolation of I ⁇ Ip may be found in US20110190194 , which describes the use of DEAE chromatography with a low pH buffer, in particular a pH less than 4.0 (e.g., pH 4.0, 3.7, 3.5, 3.4, 3.3, 3.1, 2.9, 2.0), to isolate I ⁇ Ip.
  • the method may involve more than one buffer step, where the subsequent buffer may have a lower pH than the first.
  • the buffer may be acetic acid, sodium acetate, citric acid, glycine, phosphate or salt buffer.
  • the first buffer may be a salt buffer with a salt concentration of 290 mM NaCl and the second buffer may have a pH of about 2.9.
  • the I ⁇ Ip-depleted blood product material may include the flow-through from a support capable of capturing I ⁇ Ip, such as a chromatography column. If a wash is applied to this support, the wash may be optionally included in the I ⁇ Ip-depleted blood product material or, alternatively, be itself an I ⁇ Ip-depleted blood product material.
  • a blood product material may be admixed with a loading buffer before being applied to a support.
  • a loading buffer of the present invention may be a buffer that permits or enhances the retention of one or more blood products on a support.
  • a loading buffer of the present invention may also, or alternatively, decrease retention of contaminants on a support.
  • a loading buffer may be a salt buffer of about 10 to about 300 mM salt, such as a salt buffer of about 10, 20, 30, 40, 50, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280 or 300 mM salt.
  • the support is a QA support and the loading buffer includes about 10 to 100 mM salt, such as 10, 20, 30, 40, 50, 60, 75, 80, 85, 90, 95, or 100 mM salt.
  • the pH is from 5.5 to 6.5, such as 5.5, 5.7, 5.9, 6.0, 6.1, 6.3, or 6.5.
  • the support is a QA support, the loading buffer includes about 50 mM salt, and the pH is about 6.0.
  • the support is a DEAE support and the loading buffer includes about 150 to 250 mM salt, such as 150, 175, 200, 225, or 250 mM salt.
  • the pH is from 7.0 to 8.0, such as 7.0, 7.2, 7.4, 7.6, 7.8, or 8.0.
  • the support is a DEAE support, the loading buffer includes about 200 mM salt, and the pH is about 7.6.
  • a blood product material admixed with a salt buffer may permit or enhance retention of I ⁇ Ip while decreasing the retention of contaminants.
  • Other examples of loading buffers are known in the art.
  • the I ⁇ Ip-depleted blood product material may have in an I ⁇ Ip family:IgG weight ratio less than about 1:30, such as about 1:40, 1:50, 1:100, 1:200, or 1:300, and one of factor VIII in a factor VIII:I ⁇ Ip family weight ratio greater than about 1:10 6 , such as about 1:10 5 , 1:10 4 , 1:10 3 , 1:10 2 , or 1:10, and von Willebrand factor in a von Willebrand factor:I ⁇ Ip family weight ratio greater than about 1:40, such as 1:30, 1:20, 1:10, 1:5, or 1:1.
  • the present invention further relates to compositions required for or derived by the isolation of one or more blood products from an I ⁇ Ip-depleted blood product material.
  • Blood products of the present invention include I ⁇ Ip, albumin, IgA, IgG, IgM, IgD, IgG, IVIg, anti-D IgG, hepatitis B IgG, measles IgG, rabies IgG, tetanus IgG, Varicella Zoster IgG, fibrinogen (factor I), prothrombin (factor II), factor III, factor V, VII, factor VIII, factor IX, factor X, factor XI, factor XII, factor XIII, fibronectin, alpha-1 antitrypsin, alpha-2 antiplasmin, urokinase, anti-thrombin III, C1-inhibitor, protein C, protein S, protein Z, protein Z-related protease inhibitor, plasminogen, tissue plasminogen activator, plasminogen activator inhibitor-1, plasminogen activator inhibitor-2, von Willebrand factor, factor H, prekallikrein, high-molecular
  • the inter-alpha inhibitor protein (I ⁇ Ip) family is a group of plasma-associated serine protease inhibitors. Members of this family are composed of heavy chain and light chain (bikunin) polypeptide subunits that are covalently linked by a glycosaminoglycan. In these heavy and light chain forms, bikunin remains inactive until its release by partial proteolytic degradation, a mechanism that serves as a means to regulate activity.
  • I ⁇ Ip may inhibit serine proteases that are involved in inflammation, e.g., elastase, plasmin and cathepsin G. I ⁇ Ip may be useful in the treatment of certain diseases and disorders, e.g., sepsis, septic shock, endotoxic shock, disseminated intravascular coagulation, and fibroproliferation.
  • Albumin is the main protein of plasma.
  • the primary biological function of albumin is to regulate the colloidal osmotic pressure of blood.
  • Albumin is capable of interacting with water, cations, fatty acids, hormones, bilirubin, thyroxine and other compounds.
  • Albumin may be used to treat patients with blood loss, shock, severe burns or other medical conditions. It can also be used as a component of cell growth media or as an excipient for pharmacologically active compounds.
  • Immunoglobulins are endogenous proteins which circulate in the blood and perform diverse functions. They are critical to immune function. Immunoglobulins are composed of four polypeptide chains, two light chains and two heavy chains. Immunoglobulin types are determined by the heavy chain, and include IgM, IgD, IgG, IgE and IgA. Immunoglobulins may be further defined by their specific compositions or functions.
  • Clotting factors are blood proteins that control bleeding by directing the clotting process. Circulating clotting factors are inactive, but injury initiates a coagulation cascade. The clotting process involves the contraction of blood vessels near a damaged area, followed by an accumulation of platelets. The platelets release chemical signals that result in the formation of a platelet plug. On the platelet surface, clotting factors form a fibrin clot.
  • Clotting factors include factors I (fibrinogen), II (prothrombin), III (tissue factor), IV (calcium), V (labile factor), VII (stable factor), VIII (antihemophilic factor A), IX (antihemophilic factor B), X (Stuart Prower factor), XI (antihemophilic factor C), XII (Hageman factor), and XIII (fibrin stabilizing factor).
  • Fibrinogen is a plasma glycoprotein converted to fibrin by thrombin in the presence of calcium ions. Most of the fibrinogen found in blood is synthesized in the liver. During clotting, fibrin threads form a cross-linked meshwork that contributes to the formation of a blood clot. Levels of fibrinogen increase in association with inflammation, hemostatic stress, pregnancy and other medical conditions.
  • Tissue factor is a cell surface glycoprotein. Tissue factor interacts with stable factor, a serine protease, catalyzing the formation of thrombin from prothrombin. Some cells release tissue factor in response to blood vessel damage.
  • Antihemophilic factor A is a glycoprotein cofactor that circulates in complex with von Willebrand factor, from which it may be released by thrombin.
  • Hageman factor a serine protease
  • antihemophilic factor C activates antihemophilic factor B.
  • antihemophilic factor B can interact with antihemophilic factor B in the presence of calcium ions and phospholipids to form a complex that activates Stuart Prower factor, a vitamin K-dependent serine protease.
  • Stuart Prower factor cleaves prothrombin to yield active thrombin, potentiating coagulation.
  • Fibrin stabilizing factor is the protein responsible for stabilizing the formation of a blood clot. Without it, blood clots form but break down, inhibiting wound healing. Fibrin stabilizing factor is a thrombin-activated transglutaminase that functions by forming amide cross links between fibrin molecules.
  • Alpha-1 antitrypsin is a protease inhibitor. Its concentration in blood may rise upon inflammation. It protects tissues from enzymes of inflammatory cells and inhibits a wide variety of proteases. For instance, it inhibits neutrophil elastase that would otherwise break down elastin and potentially result in respiratory complications such as emphysema or chronic obstructive pulmonary disease in adults or cirrhosis in children.
  • Anti-thrombin (III) is an inhibitor of the coagulation cascade. It is a protease that targets thrombin and factor X. Inhibitors of coagulation such as heparin act, in part, through the potentiation of anti-thrombin.
  • C1-inhibitor protein is a protease inhibitor that prevents spontaneous activation of the complement system. Its concentration in blood increases during inflammation. Targets may include C1r and C1s of the C1 complex of the complement pathway, MASP-1 and MASP-2 of the MBL complexes of the lectin pathway, kallikrein, FXI, FXII, and proteases of fibrinolytic, clotting, or kinin pathways.
  • the activity of C1-inhibitor may indirectly prevent the cleavage of products such as C2, C4 and MBL. Protein C may also inhibit factors V and VIII.
  • Protein C is a zymogenic serine protease that may be activated by binding of thrombin. Upon activation, protein C may proteolytically inactivate factor V and factor VIII. Protein C contributes to the regulation of blood clotting, inflammation, and cell death. It also contributes to blood vessel permeability. Because of the important role protein C plays as an anticoagulant, protein C deficiency increases the risk of thrombosis.
  • von Willebrand factor is critical to blood clotting. It is a glue-like protein that interacts with platelets to form a plug that directs blood flow at or near an injury. If this factor is lacking or abnormal, a bleeding disorder may result.
  • Factor H regulates the alternative complement pathway. It possesses three heparin-binding sites. Mutation of factor H may result in atypical hemolytic uremic syndrome, a condition in which platelets are depleted.
  • Prothrombin is a trypsin-like serine protease glycoprotein with many functions. Proteolysis of prothrombin may generate thrombin.
  • Thrombin is a protease that cleaves Arg-Gly bonds of fibrinogen, resulting in the formation of fibrin and the release of fibrinopeptides A and B. Thrombin is part of the clotting cascade and contributes to the formation of a hemostatic plug. It potentiates coagulation by activating factors V, VIII, XI and XIII. Thrombin may also contribute to anticoagulation through interaction with thrombomodulin and activation of protein C. Thrombin may also contribute to inflammation and wound healing activities, for instance by activation of neutrophils or platelets.
  • the present application describes methods for isolating one or more blood products from an I ⁇ Ip-depleted blood product material.
  • the starting blood product material for the purification of blood products may be, for example, whole plasma, cryo-poor plasma, liquid plasma, fresh frozen plasma (FFP), FFP24, frozen plasma (FP), FP24, thawed FFP, thawed FFP24, thawed FP, thawed FP24, source plasma, recovered plasma, solvent/detergent-treated plasma (SDP), platelet-rich plasma (PRP), platelet-poor plasma (PPP), serum, blood, or a diluted or concentrated preparation thereof.
  • FFP fresh frozen plasma
  • FP frozen plasma
  • FP24 FP24
  • thawed FFP thawed FFP24
  • thawed FP24 thawed FP24
  • SDP solvent/detergent-treated plasma
  • PRP platelet-rich plasma
  • PPP platelet-poor plasma
  • a blood product material may be contacted to a first support capable of retaining I ⁇ Ip.
  • a blood product material may be contacted to a DEAE chromatography column, as described in US20110190194 , or to a heparin affinity chromatography column, as described in US20120053113 .
  • the support of the present methods may be a support for a use in a method of chromatography, such as anion exchange chromatography, cation exchange chromatography, affinity chromatography, immunoaffinity chromatography, immobilized heparin chromatography, or dye-ligand chromatography.
  • Examples of chromatography devices that may be used in the methods of the present invention include DEAE columns, such as DEAE SEPHAROSE® (GE Healthcare), DEAE Ceramic HYPERD® (Pall, e.g., 20067-C001), and FRACTOGEL® EMD DEAE (Merck Millipore, 1.16888). Further examples include Heparin affinity, such as Heparin SEPHAROSE® (GE Healthcare), Heparin Hyper D (Pall, e.g., 20029-021), and TSKGEL® Heparin (Tosoh, e.g., 14444).
  • the support may be a support appropriate for nanofiltration. In some methods of the present invention, the support significantly retains the I ⁇ Ip present in the blood product material.
  • a first flow-through is collected from the first support.
  • the first flow-through is an I ⁇ Ip-depleted blood product material substantially inclusive of one or more non-lalp blood products. If the support is subsequently washed, the wash buffer flow-through may be included in the I ⁇ Ip-depleted blood product or be itself an I ⁇ Ip-depleted blood product.
  • An I ⁇ Ip-depleted blood product may comprise three or more, a majority, substantially all, or all of the non-lalp blood product that were present in the starting blood product material.
  • I ⁇ Ip may be eluted from the first support, producing a first eluate enriched with I ⁇ Ip.
  • This first eluate may also include a component of non-lalp blood products.
  • a variety of methods known in the art may be applied to further isolate I ⁇ Ip.
  • the percentage yield of I ⁇ Ip that is isolated from a blood product material may be at least about 20%, such as about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%, of the total present in the blood product material.
  • the yield of I ⁇ Ip from a blood product material may be at least about 5 ⁇ g/ml, such as about 5 ⁇ g/ml, 10 ⁇ g/ml, 20 ⁇ g/ml, 30 ⁇ g/ml, 40 ⁇ g/ml, 50 ⁇ g/ml, 100 ⁇ g/ml, 200 ⁇ g/ml, 300 ⁇ g/ml, 400 ⁇ g/ml, 500 ⁇ g/ml, 1000 ⁇ g/ml, or 1500 ⁇ g/ml.
  • the I ⁇ Ip-depleted blood product is provided.
  • At least one non-lalp blood product is isolated from the I ⁇ Ip-depleted blood product material.
  • Isolation of a non-lalp blood product may involve applying the I ⁇ Ip-depleted blood product material to a second support that captures one or more non-lalp blood products, and subsequently eluting those products from the second support.
  • Methods for isolating non-lalp blood products are known in the art. Exemplary methods are provided below.
  • Albumin may be isolated from an I ⁇ Ip-depleted blood product material by any applicable method known in the art, including precipitation, filtration, chromatography, liquid-solid extraction, and absorbance, or combinations thereof.
  • any applicable method known in the art including precipitation, filtration, chromatography, liquid-solid extraction, and absorbance, or combinations thereof.
  • US2011137283 describes the purification of albumin by diafiltration
  • US4156681 describes the purification of albumin by alcohol extraction
  • US4043997 describes the purification of albumin by selective absorbance with a polyhydroxy polymer.
  • US4177188 describes the purification of albumin by polyethylene glycol precipitation followed by thermocoagulation
  • US4086222 describes the purification of albumin by chromatography. Additional methods are also known in the art.
  • Immunoglobulins may be isolated from an I ⁇ Ip-depleted blood product material by any applicable method known in the art, including precipitation, filtration, chromatography, liquid-solid extraction, and absorbance, or combinations thereof.
  • USRE31268 describes the purification of immunoglobulins by fractionated precipitation
  • US20120053325 describes the purification of immunoglobulins by dye-ligand affinity chromatography
  • US4623541 describes the purification of immunoglobulins by a two-step ammonium sulfate fractionation procedure employing centrifugation and ion depletion.
  • individual immunoglobulin types are isolated by techniques with specificity for one or more of IgM, IgD, IgG, IgE and IgA, or particular immunoglobulins thereof. Additional methods are also known in the art.
  • Factor I may be isolated from an I ⁇ Ip-depleted blood product material by any applicable method known in the art, including precipitation, filtration, chromatography, liquid-solid extraction, and absorbance, or combinations thereof.
  • US7041790 describes the isolation of fibrinogen by an immobilized fibrinogen binding moiety conjugated to an affinity ligand.
  • Suzuki et al. describes the isolation of fibrinogen by affinity chromatography with adsorption to a ristocetin-agarose column.
  • Fibrinogen may also be isolated by cryoprecipitation followed by chemical precipitation using ethanol or ammonium sulfate ( Ismail, Purification of Fibrinogen from Human Plasma. Chemical & Biomolecular Engineering Theses, Dissertations, & Student Research. 2012 ). Additional methods are also known in the art.
  • Prothrombin may be isolated from an I ⁇ Ip-depleted blood product material by any applicable method known in the art, including precipitation, filtration, chromatography, liquid-solid extraction, and absorbance, or combinations thereof.
  • any applicable method known in the art including precipitation, filtration, chromatography, liquid-solid extraction, and absorbance, or combinations thereof.
  • US5143838 describes the isolation of prothrombin by anion exchange
  • US20120122179 describes the isolation of prothrombin by a deoxyribonucleic aptamer. Additional methods are also known in the art.
  • Factor V may be isolated from an I ⁇ Ip-depleted blood product material by any applicable method known in the art, including precipitation, filtration, chromatography, liquid-solid extraction, and absorbance, or combinations thereof.
  • any applicable method known in the art including precipitation, filtration, chromatography, liquid-solid extraction, and absorbance, or combinations thereof.
  • Chiu et al. describes the isolation of factor V by polyethylene glycol precipitation followed by an immunoaffinity column ( Chiu et al, J. Clin Invest. 72: 493-503, 1983 )
  • Esnouf and Jobin describe the isolation of factor V by adsorption to a phosphorylated cellulose column optionally followed by ultrafiltration ( Esnouf and Jobin, Biochem. J. 102: 660-665, 1967 ). Additional methods are also known in the art.
  • Factor VII may be isolated from an I ⁇ Ip-depleted blood product material by any applicable method known in the art, including precipitation, filtration, chromatography, liquid-solid extraction, and absorbance, or combinations thereof.
  • any applicable method known in the art including precipitation, filtration, chromatography, liquid-solid extraction, and absorbance, or combinations thereof.
  • Bajaj et al. describe the isolation of Factor VII by adsorption onto barium citrate, followed by ammonium sulfate fractionation, DEAE-SEPHADEX® chromatography and preparative polyacrylamide gel electrophoresis ( Bajaj et al., J. Biol. Chem. 256: 253-259, 1981 ).
  • Kisiel and Davie isolate factor VII by barium sulfate adsorption followed by elution DEAE-SEPHADEX® batchwise adsorption and elution, benzamide-agarose column chromatography, heparin-agarose column chromatography and preparative polyacrylamide gel disc electrophoresis ( Kisiel and Davie, Biochem. 14: 4928-4934, 1975 ).
  • US4637932 describes the isolation of factor VII using a divalent metal salt adsorbent followed by an anionic exchange resin.
  • Broze and Majerus describe the isolation of factor VII by barium citrate adsorption and elution and ammonium sulfate fractionation followed by two steps of QAE-SEPHADEX® column chromatography, SEPHADEX® G-100 column chromatography, and gel filtration on a SEPHADEX® G-25 column ( Broze and Majerus, J. Biol. Chem. 255: 1242-1247, 1980 ).
  • Hedner and Kisiel describe the isolation of factor VII by DEAE-SEPHAROSE® chromatography, ultra filtration, dialysis, QAE-SEPHADEX® A-50 chromatography, ultrafiltration, dialysis, and preparative electrophoresis ( Hedner and Kisiel, J Clin. Invest. 71: 1836-1841, 1983 ). Additional methods are also known in the art.
  • Factor VIII may be isolated from an I ⁇ Ip-depleted blood product material by any applicable method known in the art, including precipitation, filtration, chromatography, liquid-solid extraction, and absorbance, or combinations thereof.
  • US4758657 describes the isolation of factor VIII:C by adsorption onto a hydrophobic interaction matrix.
  • US4798675 describes the isolation of Factor VIII:C by adsorption onto a phospholipid coated support structure that is predominantly phosphatidylserine.
  • US4789733 describes the isolation of factor VIII by precipitation with a sulphated polysaccharide, especially heparin.
  • US5288853 describes the isolation of factor VIII complex using a heparin-coupled chromatographic medium, with further purification by precipitation with glycine and NaCl.
  • US6143179 describes the isolation of factor VIII by affinity-chromatography with immobilized cellular von-Willebrand factor or a derivative thereof.
  • US5259951 describes the isolation of factor VIII by ion exchange column chromatography.
  • EP0317279 and US4361509 describe the isolation of factor VIII by immunoaffinity.
  • US4758657 describes the isolation of factor VIII:C using a hydrophobic interaction matrix.
  • US5245014 describes the isolation of factor VIII by gel filtration chromatography under group separation conditions. Additional methods are also known in the art.
  • Factor IX may be isolated from an I ⁇ Ip-depleted blood product material by any applicable method known in the art, including precipitation, filtration, chromatography, liquid-solid extraction, and absorbance, or combinations thereof.
  • US5457181 describes the isolation of factor IX by DEAE-SEPHADEX® chromatography followed successively by ion-exchange chromatography on DEAE-SEPHAROSE® and affinity chromatography on heparin-SEPHAROSE®.
  • US5919909 describes the isolation of factor IX by a precipitation step, preferably using ammonium sulfate, leaving factor IX in the supernatant, from which it is further purified by chromatography.
  • Factor IX may also be isolated by a monoclonal antibody column, as described in US6732716 . Additional methods are also known in the art.
  • Factor X may be isolated from an I ⁇ Ip-depleted blood product material by any applicable method known in the art, including precipitation, filtration, chromatography, liquid-solid extraction, and absorbance, or combinations thereof.
  • US5378365 describes the isolation of factor X by repeated ion exchange chromatographic separations followed by adsorption chromatography on metal ions. Bajaj et al.
  • Factor XI may be isolated from an I ⁇ Ip-depleted blood product material by any applicable method known in the art, including precipitation, filtration, chromatography, liquid-solid extraction, and absorbance, or combinations thereof.
  • any applicable method known in the art including precipitation, filtration, chromatography, liquid-solid extraction, and absorbance, or combinations thereof.
  • US20100062512 describes the isolation of factor XI by hydrophobic charge induction chromatography.
  • US5252217 isolates factor XI by a filtration-adsorption step and a single step of chromatography on cation exchange resin. Additional methods are also known in the art.
  • Factor XII may be isolated from an I ⁇ Ip-depleted blood product material by any applicable method known in the art, including precipitation, filtration, chromatography, liquid-solid extraction, and absorbance, or combinations thereof.
  • Takahashi and Saito describe the isolation of factor XII by monoclonal antibody-immunoaffinity column chromatography followed by gel filtration ( Takahashi and Saito, J. Biochem. 103: 641-643, 1988 ).
  • Robin and Colman describe the isolation of factor XII by ammonium sulfate fractionation, two zinc chelate SEPHAROSE® affinity chromatography steps and gel filtration ( Robin and Colman, Thrombosis Res. 41: 89-98, 1986 ).
  • Chan and Movat describe the isolation of factor XII by adsorption with aluminum hydroxide, precipitation with polyethylene glycol, anion exchange chromatography on QAE- and a final step of either gel filtration on SEPHADEX® G-100 or affinity chromatography on an immunoadsorbent column ( Chan and Movat, Thrombosis Res. 8: 337-349, 1976 ). Additional methods are also known in the art.
  • Factor XIII may be isolated from an I ⁇ Ip-depleted blood product material by any applicable method known in the art, including precipitation, filtration, chromatography, liquid-solid extraction, and absorbance, or combinations thereof.
  • US5047506 describes the isolation of factor XIII by affinity chromatography.
  • US20080176789 describes the isolation of a factor XIII polypeptide by sequential anion exchange chromatography and hydrophobic interaction chromatography.
  • US5688919 describes the isolation of factor XIII by immunoaffinity chromatography.
  • US20080281080 describes the isolation of factor XIII by immobilized metal affinity chromatography with optional further fractionation by various chromatography methods.
  • US5204447 describes the isolation of factor XIII by precipitation by adjusting the pH of a biological fluid to about pH 5.5 to 6.5 and recovering the precipitated factor XIII. Additional methods are also known in the art.
  • Alpha-1 antitrypsin may be isolated from an I ⁇ Ip-depleted blood product material by any applicable method known in the art, including precipitation, filtration, chromatography, liquid-solid extraction, and absorbance, or combinations thereof.
  • any applicable method known in the art including precipitation, filtration, chromatography, liquid-solid extraction, and absorbance, or combinations thereof.
  • US20090292114 describes the purification of alpha-1 antitrypsin by at least two metal chelate chromatography steps and CN101274956 describes the purification of alpha-1 antitrypsin by precipitation, gel chromatography, and ultrafiltration. Additional methods are also known in the art.
  • Anti-thrombin (III) may be isolated from an I ⁇ Ip-depleted blood product material by any applicable method known in the art, including precipitation, filtration, chromatography, liquid-solid extraction, and absorbance, or combinations thereof.
  • US3842061 describes the purification of anti-thrombin (III) by adsorption onto a water-insoluble gel matrix comprised primarily of cross-linked sulfated carbohydrate
  • US4510084 describes the purification of anti-thrombin (III) by interaction with heparin or heparinoid followed by adsorption with an anion exchanger. Additional methods are also known in the art.
  • C1-inhibitor protein may be isolated from an I ⁇ Ip-depleted blood product material by any applicable method known in the art, including precipitation, filtration, chromatography, liquid-solid extraction, and absorbance, or combinations thereof.
  • any applicable method known in the art including precipitation, filtration, chromatography, liquid-solid extraction, and absorbance, or combinations thereof.
  • US5030578 describes the purification of C1-inhibitor protein by PEG fractionation, jacalin-agarose chromatography and hydrophobic interaction chromatography on phenyl-SEPHAROSE®.
  • US07/815870 describes the isolation of C1-inhibitor protein by antibody capture. Additional methods are also known in the art.
  • Von Willebrand factor may be isolated from an I ⁇ Ip-depleted blood product material by any applicable method known in the art, including precipitation, filtration, chromatography, liquid-solid extraction, and absorbance, or combinations thereof.
  • any applicable method known in the art including precipitation, filtration, chromatography, liquid-solid extraction, and absorbance, or combinations thereof.
  • US5854403 describes the isolation of von Willebrand factor by quaternary amino anion exchange.
  • US7939643 describes the isolation of von Willebrand factor by hydroxylapatite chromatography. Additional methods are also known in the art.
  • Factor H may be isolated from an I ⁇ Ip-depleted blood product material by any applicable method known in the art, including precipitation, filtration, chromatography, liquid-solid extraction, and absorbance, or combinations thereof.
  • any applicable method known in the art including precipitation, filtration, chromatography, liquid-solid extraction, and absorbance, or combinations thereof.
  • US20120053113 describes the purification of factor H by cryoprecipitation and anion exchange. Additional methods are also known in the art.
  • Thrombin may be isolated from an I ⁇ Ip-depleted blood product material by any applicable method known in the art, including precipitation, filtration, chromatography, liquid-solid extraction, and absorbance, or combinations thereof.
  • US4965203 describes the purification of thrombin with a DEAE agarose column.
  • thrombin may be produced from prothrombin that has been isolated from an I ⁇ Ip-depleted blood product material.
  • US5393666 and US5677162 describe the treatment of prothrombin with calcium ions to yield thrombin and US5151355 describes the treatment of prothrombin with thromboplastin in the presence of calcium, followed by filtration, an anion-exchange agarose column, and a cation-exchange agarose column to yield thrombin.
  • US5432062 describes the treatment of prothrombin with proteases in the presence of a detergent or certain chaotropic substances to produce thrombin. Additional methods are also known in the art.
  • Isolation of a blood product may yield at least about 10%, such as about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, of the total amount of that blood product present in the starting blood product material.
  • the yield of a particular blood product isolated from a blood product material will depend, in part, upon the quantity of that blood product present in the blood product starting material.
  • the yield of a blood product isolated from a blood product material may be at least about 1 pg/ml starting material, such as about 1 pg/ml, 5 pg/ml, 10 pg/ml, 20 pg/ml, 30 pg/ml, 40 pg/ml, 50 pg/ml, 60 pg/ml, 70 pg/ml, 80 pg/ml, 100 pg/ml, 200 pg/ml, 300 pg/ml, 400 pg/ml, 500 pg/ml, or 1 ng/ml.
  • the yield of a blood product isolated from a blood product material may be at least about 5 ng/ml starting material, such as about 10 ng/ml, 20 ng/ml, 30 ng/ml, 40 ng/ml, 50 ng/ml, 60 ng/ml, 70 ng/ml, 80 ng/ml, 100 ng/ml, 200 ng/ml, 300 ng/ml, 400 ng/ml, 500 ng/ml, or 1 ⁇ g/ml.
  • the yield of a blood product isolated from a blood product material may be at least about 5 ⁇ g/ml starting material, such as about 10 ⁇ g/ml, 20 ⁇ g/ml, 30 ⁇ g/ml, 40 ⁇ g/ml, 50 ⁇ g/ml, 60 ⁇ g/ml, 70 ⁇ g/ml, 80 ⁇ g/ml, 90 ⁇ g/ml, 100 ⁇ g/ml, 200 ⁇ g/ml, 300 ⁇ g/ml, 400 ⁇ g/ml, or 500 ⁇ g/ml, 1 mg/ml, 2mg/ml, 3 mg/ml, 4 mg/ml, 5 mg/ml, 6 mg/ml, 7 mg/ml, 8 mg/ml, 9 mg/ml, 10 mg/ml, 20 mg/ml, 30 mg/ml, 40 mg/ml, 50 mg/ml, 60 mg/ml, 70mg/ml, 80 mg/ml, 90 mg
  • compositions that may be produced through the described methods.
  • One of these compositions is an I ⁇ Ip-depleted blood product material.
  • Isolated blood products are known to treat particular medical conditions. Blood products isolated by the methods of the present invention, including I ⁇ Ip, may be useful in the treatment of such conditions. Pharmaceutical compositions of blood products isolated by the methods of the present invention may be administered or provided to a subject in need in a pharmaceutically acceptable dosage.
  • Von Willebrand factor is isolated from I ⁇ Ip-depleted FFP24.
  • FFP24 is thawed and diluted 1:10 in plasma dilution buffer (25 mM Tris, 200 mM NaCl, pH 7.6) and applied to a DEAE monolithic column.
  • plasma dilution buffer 25 mM Tris, 200 mM NaCl, pH 7.6
  • the flow-through is collected and additional plasma dilution buffer is applied to allow the starting material to pass through the column completely.
  • the additional plasma dilution buffer may then be included with the flow-through.
  • the flow-through peak returns to baseline, the column is washed with low pH buffer (150 mM Acetic acid, pH 4.0, or 200 mM Acetic acid, pH 3.3) and the peak is collected.
  • the column is further washed with a higher pH buffer (100 mM Tris, 100 mM NaCl, pH 7.6) to restore the pH.
  • Bound protein is eluted with a high salt elution buffer (25 mM Tris, 1000 mM NaCl, pH 7.6).
  • the peak is collected and this fraction contains highly pure I ⁇ Ip.
  • I ⁇ Ip may then be further purified to exchange buffer and remove low molecular weight solutes and salts by ultrafiltration or diafiltration using a membrane cut off of 30 kDa.
  • Von Willebrand factor is isolated from the flow-through.
  • the flow-through is filtered over an anion exchanger column (EMD-TMAE-FRACTOGEL® (Merck)) that has been equilibrated with buffer (20 mM Tris-HCI, pH 7.4). Subsequently, the column is washed with additional buffer. Foreign materials are removed by washing the column with 200 mM NaCl buffer. The Von Willebrand factor is then eluted from the column with another buffer (280 mM NaCl 20 mM Tris-HCI, pH 7.4). Subsequently, residual material, which is possibly present, is eluted from the column with 1M NaCl.
  • albumin is isolated from I ⁇ Ip-depleted cryo-poor plasma.
  • Cryo-poor plasma is diluted 1:10 in dilution buffer (40 mM Tris, 200 mM NaCl, pH 7.6) and applied to a DEAE monolithic column.
  • the flow-through is collected and additional buffer (25 mM Tris, 200 mM NaCl, pH 7.6) is applied to the column to allow the starting material to pass through the column completely.
  • the additional buffer may then be included with the first flow-through.
  • salt-containing wash buffer 40 mM Tris-HCI, 290 mM NaCl, pH 7.6
  • the column is additionally washed with low pH buffer (200 mM Na-Acetate, pH 2.95) and the peak is collected.
  • low pH buffer 200 mM Na-Acetate, pH 2.905
  • bound protein is eluted with high salt elution buffer (40 mM Na-Citrate, 1000 mM NaCl, pH 6.50). The peak is collected; this fraction contains highly pure I ⁇ Ip.
  • Albumin is isolated from the flow-through.
  • the flow-through is applied to a DEAE-SEPHADEX® A-50 (DEAE-substituted cross-linked dextran) that has been allowed to swell in 0.075M NaCl solution and has been decanted 3 times, autoclaved at 121 °C for 0.5 hours, washed with 1M NaCl, and suspended in 0.075M NaCl. The suspension is stirred for 45 minutes and then the DEAE-SEPHADEX® A-50 gel is filtered off, whereas the filtrate is frozen and stored at -20°C.
  • DEAE-SEPHADEX® A-50 DEAE-substituted cross-linked dextran
  • This frozen suspension is thawed at +4°C and adjusted to pH 8.0 with 0.5M NaOH solution, after which polyethylene glycol 4000 (MW 3000 - 3700) is added to the pH adjusted plasma fraction.
  • polyethylene glycol 4000 MW 3000 - 3700
  • the precipitate is removed by centrifugation at 1800 g for 10 minutes at +4°C.
  • the supernatant is adjusted to pH 4.8 with 0.5M HCl at +4°C, and additional polyethylene glycol 4000 is added to a final concentration of 22% (w/v).
  • the mixture is stirred at +4°C for 30 minutes and the albumin containing precipitate is collected by centrifugation at 1800 g for 10 minutes at +4°C.
  • the precipitate is dissolved at +4°C in distilled water and pH is adjusted to 7.0 with 0.5M NaOH.
  • the solution contains the albumin. Further purification of albumin is optionally achieved by application to an anion exchanger followed by application to a cation exchanger.
  • Alpha-1 antitrypsin is isolated from I ⁇ Ip-depleted whole plasma.
  • Whole plasma is diluted 1:10 in dilution buffer (40 mM Tris, 200 mM NaCl, pH 7.6) and applied to a DEAE monolithic column.
  • the flow-through is collected and additional buffer (25 mM Tris, 200 mM NaCl, pH 7.6) is applied to the column to allow the starting material to pass through the column completely.
  • the additional buffer may then be included with the first flow-through.
  • salt-containing wash buffer 40 mM Tris-HCI, 290 mM NaCl, pH 7.6
  • the column is additionally washed with low pH buffer (200 mM Na-Acetate, pH 2.95) and the peak is collected.
  • low pH buffer 200 mM Na-Acetate, pH 2.905
  • bound protein is eluted with high salt elution buffer (40 mM Na-Citrate, 1000 mM NaCl, pH 6.50). The peak is collected; this fraction contained highly pure I ⁇ Ip.
  • the flow-through is frozen and subjected to a controlled thaw at -0.5°C to 2°C during which some proteins precipitate.
  • the supernatant is collected, treated with CELITE®, and then filtered to remove unwanted proteins.
  • the resulting supernatant is adjusted to a pH of 5.85 with acetate buffer and ethanol is added to 17-21% v/v.
  • the temperature of the ensuing precipitation is maintained between -4°C and -6°C, such that the precipitate includes Fraction 1 and precipitate A of the Kistler and Nitschmann process (ibid).
  • the supernatant is diluted 1:1 with buffer (10 mM NaH 2 PO 4 , 10 mM NaOH, pH 11).
  • the pH of the resulting solution is between 6 and 7 with conductivity less than 7 mS/cm. pH is reduced to between 5.5 and 6.5 with dilute acetic acid just prior to loading onto a Capto Q SEPHAROSE® column equilibrated with buffer (20 mM phosphate, 30 mM NaCl, pH 6.2). Alpha-1 antitrypsin is then eluted with buffer (20 mM phosphate containing 170 mM NaCl, pH 6.2). 2.5 mM of imidazole is added to the Alpha-1 antitrypsin fraction eluted from the Capto Q column, which is then loaded onto a HisTrap column stripped of its nickel ions and re-charged with divalent copper cations. At this imidazole concentration and using this type of chelating solid support, some contaminants, but not Alpha-1 antitrypsin, bind to the solid support. The flow-through thus contains Alpha-1 antitrypsin.
  • a polysorbate 20/tri-n-butyl phosphate mixture is added according to EP-A 0131740 .
  • the solvent detergent (SD) treated Alpha-1 antitrypsin fraction is loaded onto a chelating SEPHAROSE® solid support (iminodiacetic acid chelating ligand) charged with copper.
  • a chelating SEPHAROSE® solid support iminodiacetic acid chelating ligand
  • the Alpha-1 antitrypsin is bound by the solid support whilst contaminants are not.
  • the Alpha-1 antitrypsin is then eluted with 10 mM imidazole solution.

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Claims (12)

  1. Verfahren zur Isolierung eines oder mehrerer Blutprodukte aus einem Inter-alpha-Inhibitorprotein (IαIp)-abgereicherten Blutproduktmaterial, umfassend:
    (a) das chromatographische Abreichern von wenigstens etwa 80% IαIp aus einem Blutproduktmaterial, das ausgewählt ist aus der Gruppe, bestehend aus Vollplasma, kryoarmem Plasma, flüssigem Plasma, gefrorenem Frischplasma (GFP), GFP24, gefrorenem Plasma (FP), FP24, aufgetautem GFP, aufgetautem GFP24, aufgetautem FP, aufgetautem FP24, Quellplasma, rückgewonnenem Plasma, Lösungsmittel/Detergens-behandeltem Plasma (SDP), plättchenreichem Plasma (PRP), plättchenarmem Plasma (PPP), Serum, Blut, und einem verdünnten oder konzentrierten Präparat davon, um das IαIp-abgereicherte Blutproduktmaterial herzustellen,
    wobei das IαIp-abgereicherte Blutproduktmaterial drei oder mehr Blutprodukte umfasst, die ausgewählt sind unter IVIg, Alpha-1-Antitrypsin, C1-Inhibitor, Albumin, IgA, IgG, IgM, IgD, IgG, Anti-D-IgG, Hepatitis-B-IgG, Masern-IgG, Tollwut-IgG, Tetanus-IgG, Varicella-Zoster-IgG, Fibrinogen (Faktor I), Prothrombin (Faktor II), Thrombin, Antithrombin III, Faktor III, Faktor V, Faktor VII, Faktor VIII, Faktor IX,
    Faktor X, Faktor XI, Faktor XII, Faktor XIII, Fibronektin, Alpha2-Antiplasmin, Urokinase, Protein C, Protein S, Protein Z, dem Protein Z-abhängigen Proteaseinhibitor, Plasminogen, Gewebe-Plasminogenaktivator, Plasminogenaktivator-Inhibitor-1, Plasminogenaktivator-Inhibitor-2, von-Willebrand-Faktor, Faktor H, Präkallikrein, hochmolekularem Kininogen und Heparin-Cofaktor II, und
    (b) das chromatographische Isolieren eines oder mehrerer der Blutprodukte aus dem IαIp-abgereicherten Blutproduktmaterial durch Kontaktieren des IαIp-abgereicherten Blutproduktmaterials mit einem Träger, so dass eines oder mehrere der Blutprodukte im Wesentlichen auf dem Träger zurückbehalten wird.
  2. Verfahren nach Anspruch 1, wobei das IαIp-abgereicherte Blutproduktmaterial im Wesentlichen 10 oder mehr oder 20 oder mehr Nicht-IαIp-Blutprodukte umfasst, die ausgewählt sind unter IVIg, Alpha-1-Antitrypsin, C1-Inhibitor, Albumin, IgA, IgG, IgM, IgD, IgG, Anti-D-IgG, Hepatitis-B-IgG, Masern-IgG, Tollwut-IgG, Tetanus-IgG, Varicella-Zoster-IgG, Fibrinogen (Faktor I), Prothrombin (Faktor II), Thrombin, Antithrombin III, Faktor III, Faktor V, Faktor VII, Faktor VIII, Faktor IX, Faktor X, Faktor XI, Faktor XII, Faktor XIII, Fibronektin, Alpha2-Antiplasmin, Urokinase, Protein C,
    Protein S, Protein Z, dem Protein Z-abhängigen Proteaseinhibitor, Plasminogen, Gewebe-Plasminogenaktivator, Plasminogenaktivator-Inhibitor-1, Plasminogenaktivator-Inhibitor-2, von-Willebrand-Faktor, Faktor H, Präkallikrein, hochmolekularem Kininogen und Heparin-Cofaktor II.
  3. Verfahren nach Anspruch 1 oder 2, wobei der Isolierschritt (b) die Schritte umfasst: Eluieren einer Fraktion aus dem Träger, die mit wenigstens einem der im Wesentlichen zurückbehaltenen Blutprodukte angereichert ist, gegebenenfalls wobei der Träger ein(e) Chromatographiesäule, Membran, Scheibe oder Chip ist.
  4. Verfahren nach einem der Ansprüche 1 bis 3, wobei
    (i) das IαIp-abgereicherte Blutproduktmaterial ein Blutproduktmaterial ist, das an IαI um wenigstens etwa 80% der in dem Quell-Blutproduktmaterial vorhandenen Gesamtmenge abgereichert ist, gegebenenfalls wobei das IαIp-abgereicherte Blutproduktmaterial an IαI um wenigstens etwa 90% der in dem Quell-Blutproduktmaterial vorhandenen Gesamtmenge abgereichert ist;
    (ii) das IαIp-abgereicherte Blutproduktmaterial ein Blutproduktmaterial ist, das an PαI um wenigstens etwa 80% der in dem Quell-Blutproduktmaterial vorhandenen Gesamtmenge abgereichert ist, gegebenenfalls wobei das IαIp-abgereicherte Blutproduktmaterial an PαI um wenigstens etwa 90% der in dem Quell-Blutproduktmaterial vorhandenen Gesamtmenge abgereichert ist;
    (iii) das IαIp-abgereicherte Blutproduktmaterial ein Blutproduktmaterial ist, das an Bikunin um wenigstens etwa 80% der in dem Quell-Blutproduktmaterial vorhandenen Gesamtmenge abgereichert ist, gegebenenfalls wobei das IαIp-abgereicherte Blutproduktmaterial an Bikunin um wenigstens etwa 90% der in dem Quell-Blutproduktmaterial vorhandenen Gesamtmenge abgereichert ist;
    (iv) das IαIp-abgereicherte Blutproduktmaterial ein Blutproduktmaterial ist, das an zwei oder mehr IαIp-Familienmitgliedern um wenigstens etwa 80% der in dem Quell-Blutproduktmaterial vorhandenen Gesamtmenge abgereichert ist, gegebenenfalls wobei das IαIp-abgereicherte Blutproduktmaterial an zwei oder mehr IαIp-Familienmitgliedern um wenigstens etwa 90% der in dem Quell-Blutproduktmaterial vorhandenen Gesamtmenge abgereichert ist, oder
    (v) das IαIp-abgereicherte Blutproduktmaterial ein Blutproduktmaterial ist, das an IαI und PαI um wenigstens etwa 80% der in dem Quell-Blutproduktmaterial vorhandenen Gesamtmenge abgereichert ist, gegebenenfalls wobei das lalp-abgereicherte Blutproduktmaterial an IαI und PαI um wenigstens etwa 90% der in dem Quell-Blutproduktmaterial vorhandenen Gesamtmenge abgereichert ist.
  5. Verfahren nach einem der Ansprüche 1 bis 4, wobei das Blutproduktmaterial IαIp und IgG in einem Gewichtsverhältnis von IαIp-Familie-IgG von etwa 1:30, etwa 1:5 oder zwischen etwa 1:30 und etwa 1:5, und einen der Faktoren Faktor VIII in einem Gewichtsverhältnis von Faktor VIII:IαIp-Familie kleiner oder gleich etwa 1:106 und von-Willebrand-Faktor in einem Gewichtsverhältnis von von-Willebrand-Faktor-IαIp-Familie kleiner oder gleich etwa 1:40 umfasst.
  6. Verfahren nach einem der Ansprüche 1 bis 5, wobei das Blutproduktmaterial vor dem Schritt (a) mit Ladepuffer vermischt wird,
    gegebenenfalls wobei der Ladepuffer etwa 10 bis etwa 300 mM Salz umfasst, gegebenenfalls wobei der Ladepuffer etwa 50 bis etwa 250 mM Salz umfasst, gegebenenfalls wobei der Ladepuffer etwa 200 mM Salz umfasst.
  7. Verfahren nach einem der Ansprüche 1 bis 6, wobei das IαIp-abgereicherte Blutproduktmaterial drei oder mehr, gegebenenfalls zehn oder zwanzig oder mehr Nicht-IαIp-Blutprodukte in einer Menge größer oder gleich etwa 20% der Menge jedes der in dem Blutproduktmaterial vorhandenen Nicht-IαIp-Blutprodukte umfasst, und wobei die Nicht-IαIp-Blutprodukte ausgewählt sind unter IVIg, Alpha-1-Antitrypsin, C1-Inhibitor, Albumin, IgA, IgG, IgM, IgD, IgG, Anti-D-IgG, Hepatitis-B-IgG, Masern-IgG, Tollwut-IgG, Tetanus-IgG, Varicella-Zoster-IgG, Fibrinogen (Faktor I), Prothrombin (Faktor II), Thrombin, Antithrombin III, Faktor III, Faktor V, Faktor VII, Faktor VIII, Faktor IX, Faktor X, Faktor XI, Faktor XII, Faktor XIII, Fibronektin, Alpha2-Antiplasmin, Urokinase, Protein C, Protein S, Protein Z, dem Protein Z-abhängigen Proteaseinhibitor, Plasminogen, Gewebe-Plasminogenaktivator, Plasminogenaktivator-Inhibitor-1, Plasminogenaktivator-Inhibitor-2, von-Willebrand-Faktor, Faktor H, Präkallikrein, hochmolekularem Kininogen und Heparin-Cofaktor II.
  8. Verfahren nach einem der Ansprüche 1 bis 7, wobei Schritt (a) oder Schritt (b) das Kontaktieren des Blutproduktmaterials bzw. des IαIp-abgereicherten Blutproduktmaterials mit einer Chromatographiesäule umfasst;
    gegebenenfalls wobei Schritt (a) das Kontaktieren des Blutproduktmaterials mit einer Anionenaustauschersäule, wie einer DEAE- oder QA-Säule, umfasst.
  9. Verfahren nach einem der Ansprüche 1 bis 8, wobei Schritt (a) umfasst: das Kontaktieren des Blutproduktmaterials mit einem ersten Träger, wobei wenigstens etwa 80% des IαIp aus dem Blutproduktmaterial auf dem Träger zurückbehalten werden, und das Eluieren des zurückbehaltenen IαIp aus dem ersten Träger, wodurch ein erstes Eluat hergestellt wird, wobei das erste Eluat mit dem zurückbehaltenen IαIp angereichert ist.
  10. Verfahren nach Anspruch 9, wobei das erste Eluat aus isoliertem IαIp besteht.
  11. Verfahren nach Anspruch 9, ferner umfassend das Abtrennen des zurückbehaltenen IαIp aus dem ersten Eluat, um ein isoliertes IαIp herzustellen, gegebenenfalls wobei:
    (a) die Ausbeute an isoliertem IαIp wenigstens 5 µg/ml Blutproduktmaterial, insbesondere wenigstens 50, 100, 300, 600 oder 900 µg/ml Blutproduktmaterial, beträgt;
    (b) die Reinheit des isolierten IαIp wenigstens 5%, insbesondere wenigstens 25%, 50%, 75% oder 100%, beträgt;
    (c) das isolierte IαIp IαI, PαI oder Bikunin ist;
    (d) das isolierte IαIp zwei oder mehr lalp-Familienmitglieder, insbesondere IαI und PαI, umfasst;
    (e) das zurückbehaltene IαIp IαI, PαI oder Bikunin ist, und/oder
    (f) das zurückbehaltene IαIp zwei oder mehr IαIp-Familienmitglieder, insbesondere IαI und PαI, umfasst.
  12. Verfahren nach einem der Ansprüche 1 bis 11, wobei die Ausbeute des einen oder der mehreren in Schritt (b) isolierten Nicht-IαIp-Blutprodukte wenigstens 20%, gegebenenfalls wenigstens 50% oder wenigstens 80%, der in dem IαIpabgereicherten Blutproduktmaterial vorhandenen Gesamtmenge an Nicht-IαIp-Blutprodukten beträgt.
EP14740523.7A 2013-01-18 2014-01-17 Verfahren zur isolierung von blutprodukten aus einem inter-alpha-inhibitorprotein-abgereicherten blutproduktmaterial Revoked EP2945962B1 (de)

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WO2014039987A2 (en) * 2012-09-09 2014-03-13 Prothera Biologics, Inc. Treatment of disease using inter-alpha inhibitor proteins
WO2018053029A1 (en) 2016-09-13 2018-03-22 Prothera Biologics, Inc. Methods for treating pulmonary disease using inter-alpha inhibitor proteins
CN108144586B (zh) * 2016-12-05 2020-07-17 中国水产科学研究院黄海水产研究所 对氨基苯甲脒仿生亲和配基的仿生亲和纯化方法
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HK1217721A1 (en) 2017-01-20
ES2736283T3 (es) 2019-12-27
EP2945962A4 (de) 2016-11-02
EP2945962A1 (de) 2015-11-25
US20150361127A1 (en) 2015-12-17
AU2014207447B2 (en) 2018-11-15
AU2014207447A1 (en) 2015-09-03
WO2014113659A1 (en) 2014-07-24
CN105209477A (zh) 2015-12-30
CA2921692A1 (en) 2014-07-24

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